| Objectives To observe change of the autophagy associated protein LC3,Beclin1 and NF-κB,TNF-α in intracerebral hemorrhage rat model,In order to explore the effect of autophagy on the secondary inflammatory injury of neurons in brain tissue of intracerebral hemorrhage rat and the intervention of 3MA and to investigate the protective effect and possible mechanism of autophagy inhibition on intracerebral hemorrhage rats.Methods 1 Model preparation and grouping: fresh non-anticoagulant blood was injected into the right basal ganglia of rats to establish intracerebral hemorrhage model.180 adult male SD rats were randomly divided into the following 3 groups: sham operation group(Sham group);intracerebral hemorrhage model group(ICH model group): ICH model was made by injecting fresh non-anticoagulant blood into the right basal ganglia of rats;ICH model+inhibitor intervention group(ICH+3MA group): recieved 3MA by intraperitoneal injection at 5mg/kg 15 minutes before modeling,and then once a day.Each group was divided into five subgroups according to different time points(0.5d,1d,2d,3d,5d).2 Detection indexes and methods: Longa scoring method was used to measure behavioral score of rats;hematoxylin eosin staining(HE staining)was used to observe the pathological changes of brain tissue;Nissl staining was used to observe the pathological changes of brain tissue and count the masculine cellls;The dry-wet weight method was used to measure brain water content;transmission electron microscope was used to observe autophagic bodies;immunofluorescence confocal detection was used to detect the localization of NF-κB,TNF-α,LC3 and Beclin1;detect LC3,Beclin1,NF-κB and TNF-α by immunohistochemistry;Western blot was used to detect the expression of Beclin1,NF-κB,TNF-α.Results 1 Compared with sham group,the neurological scores of ICH model group and ICH+3MA group were significantly higher(P<0.05),and at different observation time points,there were different degrees of function deficits,such as decreased activity,sluggish response,inflexibility and even paralysis,compared with ICH model group,the neurological score of ICH+3MA group was lower(P<0.05).2 HE staining showed that the brain tissue structure of sham group was complete,the cells had no obvious pathological change and present distinctive boundary,regular arrangement,clear nuclei.In ICH model group and ICH+3MA group,there were edema around hematoma,nerve cells swelling,pyknosis or fragmentation of nuclei,necrosis and disappearance of some nerve cells,and obviously widened intercellular space.3 Nissl staining showed,compared with sham group,ICH model group present wider intercellular space more significant contracted cytoplasm and Nissl body loss(P<0.05).After 3MA intervention,the Nissl body loss of nerve cells improved significantly(P<0.05).4 The brain water contents in sham group were coincident approximately at different observation time points,and there was no brain edema.While the brain water contents rose 0.5d after the operation in ICH model group and ICH+3MA group,and gradually increase over time.Compared with sham group,the difference of brian water contents at different time points was statistically significant(P<0.05);compared with ICH group,the brain water contents decreased at different time points after 3MA intervention,and the difference was statistically significant(P<0.05).5 Transmission electron microscope observed that the autophagy bodies of ICH model group and ICH+3MA group were significantly increased compared with sham group.6 The results of immunofluorescence confocal analysis showed that NF-κB and TNF-α,LC3,Beclin1 could be co-localized separately.7 The results of immunohistochemistry showed that the expression of LC3,Beclin1,NF-κB,TNF-α in sham group was only weakly positive;compared with sham group,the expression of LC3,Beclin1,NF-κB,TNF-α in ICH model group and ICH+3MA group was significantly different.In the ICH model group,the expression of LC3 and Beclin1 increased significantly at 0.5d,peaked at 1d,and lasted to 3d,then decreased gradually;the expression of NF-κB and TNF–α increased gradually over time,peaked at 3d,and then decreased;after 3MA intervention,the trend of LC3,Beclin1,NF-κB and TNF-α was the same as that of the ICH model group,but the expression decreased.8 the results of immunoprotein imprinting showed that Beclin1,NF-κB and TNF-α in sham group were lower,was similar with physiological level.Compared with sham group,the expression of NF-κB was significantly increased in ICH group,and decreased after 3MA intervention,these differences were statistically significant(P<0.05).Compared with sham group,TNF-α protein expression in ICH model group increased significantly,and TNF-α expression decreased after 3MA intervention,the differences were statistically significant(P<0.05).The change trend of TNF-α was approximately consistent with that of NF-κB.Compared with sham group,the expression of Beclin1 in ICH model group increased significantly(P<0.05).Beclin1 decreased after 3MA intervention,compared with ICH model group,the expression of Beclin1 decreased significantly(P<0.05).Conclusions 1 Autophagy may be involved in the process of secondary inflammatory injury of neurons in peri-hematoma brain tissue in intracerebral hemorrhage rats.2 In the early stage of secondary injury,the activation of neurons autophagy in brain tissue can aggravate NF-κB-induced inflammatory response and raise TNF-α induced inflammatory damage.3 Autophagy inhibitor 3MA can inhibit autophagic activity and reduce secondary inflammatory response of neurons in brain tissue,alleviate brain injury.Figure16;Table3;Reference 143... |
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